Positional cloning and characteristics of fat-free gene

脱脂基因的定位克隆及特征

• 批准号: 6737741
• 负责人: Shiu-Ying Ho
• 金额: $ 5.05万
• 依托单位: THOMAS JEFFERSON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-07-01 至 2005-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6737741
• 关键词: artificial chromosomes; bile; biological signal transduction; cholestasis; expression cloning; gene expression; gene mutation; genetic mapping; in situ hybridization; lipid metabolism; microarray technology; postdoctoral investigator; single nucleotide polymorphism

DESCRIPTION (provided by applicant): Lipids play essential roles in all living cells, not only as key components of membranes and as a source of energy, but also as important mediators of cellular signaling. At the organismal level, individual differences in lipid metabolism can impact a variety of human diseases. In our laboratory, ethylnitrosurea (ENU) mutagenized zebrafish are screened in vivo for abnormality in lipid processing using the fluorescent reporters. One of the lethal recessive mutant, fat-free, was identified at the beginning of the large-scale screen. Biochemical analysis using fluorescent lipids analogues indicate that fat-free mutation may attenuate biliary synthesis or secretion. Although three genes (FIC1, BSEP, and MDR3) responsible for infants and children with progressive familial intrahepatic cholestasis (PFIC) are identified, it remains unknown if there are undiscovered genes that are involved in bile synthesis and/or regulation may lead to PFIC. Fat-free locus is not relevant to FIC1, BSEP or MDR3 because all three genes are mapped to different linkage groups using zebrafish radiation hybrid panel. Therefore, fat-free mutant provides an ideal potential model to discover a gene that may contribute to PFIC. The specific aims of this proposal are positionatly clone and characterization of the fat-free gene. The PCR-based analysis methods have enabled me to locate a region that is 0.06 cM (1 out of 1700 meioses) from the fat-free locus. The final stage of mapping is relied on BAC walking, SNP detection, and shotgun sequencing. To characterize fat-free gene, morpholino knock-down, genomic DNA rescue, whole mount in situ hybridization, and microarray analysis are included.

描述（由申请人提供）：脂质在所有活细胞中都起着重要作用，不仅是膜的关键组成部分和能量来源，而且是细胞信号传导的重要介体。在生物层面，脂质代谢的个体差异会影响各种人类疾病。在我们的实验室中，使用荧光记者在体内筛选乙酰硝基尿素（ENU）诱变的斑马鱼在脂质加工方面的异常。在大规模屏幕的开头发现了一个致命的隐性突变体，无脂肪的突变体。使用荧光脂质类似物的生化分析表明，无脂肪突变可能会减弱胆道的合成或分泌。尽管鉴定出了疾病性家族性肝内胆汁淤积（PFIC）的婴儿和儿童的三个基因（FIC1，BSEP和MDR3），但尚不清楚是否存在胆汁合成和/或调节的未发现的基因可能导致PFIC。无脂肪的基因座与FIC1，BSEP或MDR3无关，因为所有三个基因均使用斑马鱼辐射杂种面板映射到不同的链接组。因此，无脂肪突变体提供了理想的潜在模型，以发现可能有助于PFIF的基因。该建议的具体目的是定位克隆和无脂肪基因的表征。基于PCR的分析方法使我能够找到一个来自无脂肪基因座的0.06 cm（1700毫秒）的区域。映射的最后阶段依赖于BAC步行，SNP检测和shot弹枪测序。为了表征无脂肪的基因，含有型敲门，基因组DNA救援，整个原位杂交和微阵列分析。